ELISA optimization & Techniques
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Cell Lysis and synchronisation
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Sonication applies sound energy to agitate particles in your sample. The ultrasonic frequency used is usually greater than >20 kHz. In an experimental setting this is usually carried out using an ultrasonic bath or ultrasonic probe generally referred to as sonication.
In the laboratory sonication is used mainly as a method of cell disruption. Sonication is used to disrupt cellular membranes and release the cells contents, this process is generally referred to as sonoporation. Sonication is carried out during the preparation of protein extracts in order to break the cell apart. Although lysis buffer can be used sonication can help break the cell apart. Sonication can also be used to fragment/shear DNA,preventing it from interfering with further sample preparation. Other biological uses include the production of nanoparticles, liposomes, extraction of anthocyanins and antioxidants.
Depending on your cell type (bacterial or eukaryotic) it can be difficult to lyse certain cell types and placing them in a detergent buffer alone wont result in full cell lysis. Furthermore you may also require to lyse the cellular organelles and not just lyse the cell wall to release the cytosol. Sonication of cells using a titanium probe can help lyse cells fully and help all extract all DNA, RNA and protein contents of your cells. This can help downstream when looking for more homogenous extract for ELISA assays and immunoprecipitation.
If you follow the sonication protocol below for cell lysis you should achieve efficient lysis of your cells for your required application.
- Centrifuge cells for 5 mins at 270 x g in a microcentrifuge.
- Aspirate the remaining media and resuspend cells in 30 – 100 μL of RIPA buffer.
- Incubate the pellet on ice for 30 min.
- Sonicate the samples as follows.
- Place the sonicator probe at a frequency of 20 kHz.
- Place the cells in a 1.5 mL microcentrifuge tube and gently move under the tip of the sonnicator probe.
- The probe will begin to vibrate the buffer for 2 X 10 sec to reduce viscosity (This may result in foaming of the samples)
- In this protocol however foaming of the samples was not shown to be a problem following sonication when we carried on to immunoprecipitation, Western Blotting or ELISA assays.
- Depending on samples and viscosity of the samples, cells can be sonicated again for a further 10 s .
- Once the samples are sonicated incubate on ice for 5 min.
- Centrifuge at 10,000 x g for 20 min to pellet debris (Debris may contain unlysed cells, nuclei or unlysed organelles).
- Transfer the supernatants to a new microcentrifuge tube and label.
- Store at -20 °C.
Sarkoysl lysis of Bacterial Cells & Sonication
- Sarkosyl lysis of BL21 cells and the removal of chaperone proteins BL21 bacterial pellets were resuspended in 50 ml of ice cold Sodium Tris-EDTA (STE) buffer (10 mM Tris-HCL, pH 8.0, 1 mM EDTA, 150 mM NaCl supplemented with 100 mM PMSF).
- Add 500 ul of Lysozyme (10 mg/ ml) and incubate on ice for 15 min.
- Add 500 ul of DTT and 7 ml of Sarkosyl (10% (w/v) made up in STE buffer).
- All purification buffers must be kept ice cold and samples maintained on ice. All purification steps were carried out in the cold room where possible.
- Sonicate samples for 3 x 30 sec with a 2 min interval between eachsonication.
- Place samples on ice for further purification steps or store at -80 °C.
Hints and Tips
- Always keep your samples on ice. The energy from the sonicator which causes your sample to break apart also heats it up. If your sample get too hot the protein will start to degrade. In order to prevent this try to keep your sample on ice at all times, before, after and during if possible.
- Reduce the temperature of your sample by pulsing. Most sonicators have a pulse mode which reduces the heating up of your sample during sonication. If pulsing is not an option/not available turn the sonicator on for 5 seconds and then off. Repeat as many times as necessary.
- Dont over-Sonicate. Sonicating your sample for too long can degrade your protein. Finding that perfect balance may take some optimisation and can vary for different cell/tissue types and sample volumes.
- Sample Volume and probe size. The probe you use can vary depending on your sample size. Each probe has a recommended sample volume range. Small tips (microtips) are recommended for processing samples inside small, thin vessels and never samples larger than 50ml. Microtips are made for short processing times. Microtips will generate a considerable amount of heat in small volumes and therefore should be used in the pulse mode to prevent heat buildup.
- Protect your ears. Although sonication is ultrasonic and out of the range of human hearing the the collapse of tiny cavitation bubbles created by the sonication creates a loud screeching noise. I suggest wearing some proper ear protection, covering your ears simply wont cut it here.
- Try to limit foaming. If your probe isn’t submerged properly in your sample it can lead to foaming, however if its too deep you wont get proper lysis. If you do get some foaming dont panic, I found that foaming wasnt a problem when I carried on to immunoprecipitation, Western Blotting or ELISA assays.
- Slow and steady wins the race when it come to amplitude. As with all experiments the temptation to crank up your western to maxmium voltage or transform your cells in half the time is strong – this doesnt however always result in presentable never mind publishable results. For sonication a lower amplitude for longer will reduce heating of the sample – within reason you do have other experiments to get on with.Amplitude and intensity have a direct relationship. In order to be able to reproduce results, the amplitude setting, temperature, viscosity and volume of the sample are all parameters that need to remain consistent. The amplitude, not the power, is most critical when trying to reproduce sonication results.
- Always clean the sonicator tip between samples. Cleaning the sonicator tip is critical in limiting protein carryover. Wiping the sonicator probe with 70 %ethanol or sonication of ethanol in a beaker is an effective way of cleaning the sonicator tip.